response capability of the patients against to mitogenic, bacterial and viral ... lymphoproliferative response capability to mitogens and bacterial antigens in FA ...
Lymphoproliferative response of Fanconi anemia patients to mitogens, bacterial and viral antigens in vitro Faruk SÜZERGÖZ1,2, Ali O. GÜROL1, Fatih M. EVCİMİK1, Nevin YALMAN3 1
İstanbul Üniversitesi, Deneysel Tıp Araştırma Enstitüsü (DETAE), İmmünoloji Anabilim Dalı, İstanbul. Harran Üniversitesi, Fen-Edebiyat Fakültesi, Biyoloji Bölümü, Şanlıurfa. 3 İstanbul Üniversitesi, İstanbul Tıp Fakültesi, Tıbbi Biyoloji Anabilim Dalı, İstanbul. Türkiye 2
Summary Increased susceptibility to infections in Fanconi anemia (FA) have been investigated by determining immune response capability of the patients against to mitogenic, bacterial and viral antigens in vitro. Peripheral blood mononuclear cells (PBMC) were obtained from 11 FA patients and 11 healthy control individuals. 105 cells from each sample were assessed by using colorimetric MTT assay including 20 µg/ml pytoheamagglutinine (PHA), 10 µg/ml pokeweed mitogen (PWM), 6 µg/ml Tetanus toxoid (TT), 10 µg/ml purified protein derivative of mycobacterium (PPD) and 1.25 µg/ml cytomegalovirus (CMV) antigen. Lymphoproliferative response to PHA, PWM, TT and PPD were found significantly lower in FA patients than control group. Lymphoproliferation to CMV antigen was significantly higher in CMV seropositive than seronegative FA patients. To conclude, the lower lymphoproliferative response capability to mitogens and bacterial antigens in FA patients implied susceptibility to infections, but, it is likely in CMV seropositive FA patients, the cells encountering antigens could become immunopotent. Key words Fanconi anemia, lymphoproliferation, mitogen, cytomegalovirus Fankoni anemili hastaların mitojenlere, bakteriyel ve viral antijenlere karşı in vitro lenfoproliferatif yanıtları Özet Fankoni anemili hastaların enfeksiyonlara karşı artmış hassasiyeti, hastaların mitojenlere, bakteriyel ve viral antijenlere karşı immün cevap yetenekleri in vitro ortamda belirlenmek suretiyle incelenmiştir. Periferik kan mononükleer hücreleri (PBMC) 11 Fankoni anemili hasta ve 11 sağlıklı kontrol bireyinden alınmıştır. Her bir örnekten 105 hücre alınarak MTT yöntemiyle, 20 µg/ml fitohemaglutinin, (PHA), 10 µg/ml pokeweed mitojen (PWM), 6 µg/ml Tetanoz toksoidi (TT), 10 µg/ml saflaştırılmış mycobacterium proteini (PPD) and 1.25 µg/ml citomegalovirus (CMV) antijeni ile test edilmiştir. PHA, PWM, TT ve PPD’ ye karşı lenfoproliferatif yanıt kontrol grubuna göre Fankoni anemili hastalarda anlamlı şekilde düşük bulunmuştur. CMV seropozitif hastalarda ise CMV antijenine karşı lenfoproliferatif yanıt CMV seronegatif hastalardan daha yüksek bulunmuştur. Sonuç olarak, Fankoni anemili hastalarda mitojenlere, bakteriyel ve viral antijenlere karşı düşük lenfoproliferatif yanıtın infeksiyonlara yatkınlığın bir işareti olduğunu fakat CMV seropozitif hastalarda görüldüğü gibi antijenle temas durumunda hücrelerin immunopotent hale dönüşebildiğini söyleyebiliriz. Anahtar Kelimeler Fanconi anemisi, lenfoproliferasyon, mitojen, citomegalovirus
Introduction Fanconi Anemia (FA) is an x-linked heterogeneous autosomal recessive genetic disorder characterized by congenital abnormalities, progressive bone marrow failure, and cancer susceptibility (1, 2). Defective hematopoiesis, defective DNA repair and altered levels of certain growth factors (3, 4) in FA may affect hematopoiesis by altering the bone marrow microenvironment, leading to dysregulation of cellular homeostasis, differentiation and response to stress. (5-9) Patients with FA frequently suffer from bleeding and infection, symptoms related to thrombocytopenia and neutropenia (10). FA may include increased susceptibility to oral diseases with underlying host defense impairment coupled with periodontal infection
by cytomegalovirus (CMV) and Actinobacillus actinomycetemcomitans (11, 12). A Vibrio cholera sepsis was reported in a child with FA which was likely to be caused by the underlying hematological complications (13).
The aim of this study was to investigate the susceptibility to infections of FA patients by determining lymphoproliferative response of the peripheral blood mononuclear cells (PBMC) to specific and nonspecific stimulants in vitro.
Patients and Methods Blood samples were obtained from 11 FA patients (mean age 9.3 years; range 6 – 17) and 11 healthy individuals (mean age 8.9 years; range 3 – 13). The diagnoses of patients were confirmed by nitrogen mustard and/or diepoxybutone method to induce chromosomal Harran Üniversitesi Tıp Fakültesi Dergisi 2008;5(2):19-23 19
breakages in peripheral blood lymphocyte cultures (14). FA patients and control group individuals had primary immunization with tetanus toxoid (TT), Bacillus Calmette Guerin (BCG), diphtheria toxoid (DT), pneumococcus (Pnc), Haemophilus influenzae type b (Hib) and other childhood vaccines. Lymphocyte stimulation assay PBMC were obtained from heparinized venous blood over a Histopaque gradient (Sigma) according to the manufacturer’s instructions. The lymphocyte band (at the interface) was carefully removed and washed in RPMI 1640 medium (Sigma) supplemented with 10% (v/v) fetal calf serum, 1% L-glutamine (2.0 mM), 100 UI/ml penicillin and 100 µg/ml of streptomycin (complete medium). The assay was performed in tissue culture plates (96 wells); the wells were fulfilled with complete medium followed by 20 µg/ml phytohemagglutinin (PHA, Biochrom KG, Berlin, Germany), 10 µg/ml pokeweed mitogen (PWM, Sigma Chemical Co., St. Louis, MO, USA), 6 µg/ml tetanus toxoid (TT, Aventis Pasteur SA, Lyon, France), 10 µg/ml purified protein derivative of mycobacterium (PPD, BNCIPD Ltd. Sofia, Bulgaria), 1.25 µg/ml cytomegalovirus antigen (CMV, Abbott Lab. Abbott Park, IL) and PBMC. Culture plates were incubated at 5% CO2 and 37 oC for 72 hours prior to MTT assay. Cell proliferation was quantified by a colorimetric assay based on the reduction of [3(4,5-dimethylthyazol-2yl)-2,5-diphenyl tetrazolium] (MTT). MTT solution (5 mg/ml in RPMI 1640 medium) was added and incubated (4 hours) at the same condition. Plates were centrifuged, supernatant was removed and 100 µl of dimethyl sulphoxide, DMSO (Sigma), was added under agitation; plates were read on an ELISA reader at 540 nm using 620 nm as reference filter (Organon Technica Reader Model 530). CMV serology of FA patients CMV seropositivity for specific IgM antibodies in sera was determined with CMV IgM microparticle enzyme immunoassay (MEIA) (15) by Abbott Axsym® system (Abbott Laboratories, Diagnostics Division, Abbott Park IL). Statistical analyses Results are expressed as mean and ± standard error of the mean (SEM). Significances between FA and control groups were determined by ANOVA. Results
PHA and PWM mitogenesis In order to assess T cell function, PBMC were cultured in the presence of PHA (20 µg/ml). Cultures were assessed for proliferation by the MTT assay as shown in Figure 1. PBMC obtained from FA patients exhibited lower proliferative responses to PHA than healthy individuals. The average absorbance for control group and FA patient PBMC were 0.243 (SEM=0.008) and 0.176 (SEM=0.006), respectively, (p
